Gravitational Biology II: Interaction of Gravity with Cellular Components and Cell Metabolism [1st ed.] 978-3-030-00595-5, 978-3-030-00596-2

Table of contents :
Front Matter ....Pages i-xiv
Interaction of Gravity with Cellular Compounds (Wolfgang Hanke, Florian P. M. Kohn, Maren Neef, Rüdiger Hampp)....Pages 1-32
Interaction of Gravity with Cell Metabolism (Wolfgang Hanke, Florian P. M. Kohn, Maren Neef, Rüdiger Hampp)....Pages 33-94

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SPRINGER BRIEFS IN SPACE LIFE SCIENCES

Wolfgang Hanke Florian P.M. Kohn Maren Neef Rüdiger Hampp

Gravitational Biology II Interaction of Gravity with Cellular Components and Cell Metabolism 123

SpringerBriefs in Space Life Sciences

Series Editors Günter Ruyters Markus Braun Space Administration German Aerospace Center (DLR) Bonn, Germany

The extraordinary conditions of space, especially microgravity, are utilized for research in various disciplines of space life sciences. This research that should unravel – above all – the role of gravity for the origin, evolution, and future of life as well as for the development and orientation of organisms up to humans, has only become possible with the advent of (human) spaceflight some 50 years ago. Today, the focus in space life sciences is 1) on the acquisition of knowledge that leads to answers to fundamental scientific questions in gravitational and astrobiology, human physiology and operational medicine as well as 2) on generating applications based upon the results of space experiments and new developments e.g. in non-invasive medical diagnostics for the benefit of humans on Earth. The idea behind this series is to reach not only space experts, but also and above all scientists from various biological, biotechnological and medical fields, who can make use of the results found in space for their own research. SpringerBriefs in Space Life Sciences addresses professors, students and undergraduates in biology, biotechnology and human physiology, medical doctors, and laymen interested in space research. The Series is initiated and supervised by Dr. Günter Ruyters and Dr. Markus Braun from the German Aerospace Center (DLR). Since the German Space Life Sciences Program celebrated its 40th anniversary in 2012, it seemed an appropriate time to start summarizing – with the help of scientific experts from the various areas - the achievements of the program from the point of view of the German Aerospace Center (DLR) especially in its role as German Space Administration that defines and implements the space activities on behalf of the German government. More information about this series at http://www.springer.com/series/11849

Wolfgang Hanke • Florian P.M. Kohn • Maren Neef • Rüdiger Hampp

Gravitational Biology II Interaction of Gravity with Cellular Components and Cell Metabolism

Wolfgang Hanke Institute of Physiology University of Hohenheim Stuttgart, Germany

Florian P.M. Kohn Institute of Physiology University of Hohenheim Stuttgart, Germany

Maren Neef Institute for Microbiology and Infection Biology Tübingen (IMIT) University of Tübingen Tübingen, Germany

Rüdiger Hampp Institute for Microbiology and Infection Biology Tübingen (IMIT) University of Tübingen Tübingen, Germany

ISSN 2196-5560 ISSN 2196-5579 (electronic) SpringerBriefs in Space Life Sciences ISBN 978-3-030-00595-5 ISBN 978-3-030-00596-2 (eBook) https://doi.org/10.1007/978-3-030-00596-2 Library of Congress Control Number: 2018960184 © Springer Nature Switzerland AG 2018 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface to the Series

The extraordinary conditions in space, especially microgravity, are utilized today not only for research in the physical and materials sciences—they especially provide a unique tool for research in various areas of the life sciences. The major goal of this research is to uncover the role of gravity with regard to the origin, evolution, and future of life, and to the development and orientation of organisms from single cells and protists up to humans. This research only became possible with the advent of manned spaceflight some 50 years ago. With the first experiment having been conducted onboard Apollo 16, the German Space Life Sciences Program celebrated its 40th anniversary in 2012—a fitting occasion for Springer and the DLR (German Aerospace Center) to take stock of the space life sciences achievements made so far. The DLR is the Federal Republic of Germany’s National Aeronautics and Space Research Center. Its extensive research and development activities in aeronautics, space, energy, transport, and security are integrated into national and international cooperative ventures. In addition to its own research, as Germany’s space agency the DLR has been charged by the federal government with the task of planning and implementing the German space program. Within the current space program, approved by the German government in November 2010, the overall goal for the life sciences section is to gain scientific knowledge and to reveal new application potentials by means of research under space conditions, especially by utilizing the microgravity environment of the International Space Station (ISS). With regard to the program’s implementation, the DLR Space Administration provides the infrastructure and flight opportunities required, contracts the German space industry for the development of innovative research facilities, and provides the necessary research funding for the scientific teams at universities and other research institutes. While so-called small flight opportunities like the drop tower in Bremen, sounding rockets, and parabolic airplane flights are made available within the national program, research on the ISS is implemented in the framework of Germany’s participation in the ESA Microgravity Program or through bilateral cooperations with other space agencies. Free flyers such as BION or FOTON satellites are used in cooperation with Russia. The recently started utilization of v

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Chinese spacecrafts like Shenzhou has further expanded Germany’s spectrum of flight opportunities, and discussions about future cooperation on the planned Chinese Space Station are currently underway. From the very beginning in the 1970s, Germany has been the driving force for human spaceflight as well as for related research in the life and physical sciences in Europe. It was Germany that initiated the development of Spacelab as the European contribution to the American Space Shuttle System, complemented by setting up a sound national program. And today Germany continues to be the major European contributor to the ESA programs for the ISS and its scientific utilization. For our series, we have approached leading scientists first and foremost in Germany, but also—since science and research are international and cooperative endeavors—in other countries to provide us with their views and their summaries of the accomplishments in the various fields of space life sciences research. By presenting the current SpringerBriefs on muscle and bone physiology we start the series with an area that is currently attracting much attention—due in no small part to health problems such as muscle atrophy and osteoporosis in our modern aging society. Overall, it is interesting to note that the psycho-physiological changes that astronauts experience during their spaceflights closely resemble those of aging people on Earth but progress at a much faster rate. Circulatory and vestibular disorders set in immediately, muscles and bones degenerate within weeks or months, and even the immune system is impaired. Thus, the aging process as well as certain diseases can be studied at an accelerated pace, yielding valuable insights for the benefit of people on Earth as well. Luckily for the astronauts: these problems slowly disappear after their return to Earth, so that their recovery processes can also be investigated, yielding additional valuable information. Booklets on nutrition and metabolism, on the immune system, on vestibular and neuroscience, on the cardiovascular and respiratory system, and on psycho-physiological human performance will follow. This separation of human physiology and space medicine into the various research areas follows a classical division. It will certainly become evident, however, that space medicine research pursues a highly integrative approach, offering an example that should also be followed in terrestrial research. The series will eventually be rounded out by booklets on gravitational and radiation biology. We are convinced that this series, starting with its first booklet on muscle and bone physiology in space, will find interested readers and will contribute to the goal of convincing the general public that research in space, especially in the life sciences, has been and will continue to be of concrete benefit to people on Earth. Bonn, Germany Bonn, Germany July 2014

Günter Ruyters Markus Braun

Preface to the Series

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DLR Space Administration in Bonn-Oberkassel (DLR)

The International Space Station (ISS); photo taken by an astronaut from the space shuttle Discovery, March 7, 2011 (NASA)

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Extravehicular activity (EVA) of the German ESA astronaut Hans Schlegel working on the European Columbus lab of ISS, February 13, 2008 (NASA)

Preface

The first book on gravitational biology in our series “Springer Briefs in Space Life Sciences” entitled Gravitational Biology I—Gravity Sensing and Graviorientation in Microorganisms and Plants and published a few months ago had focused on gravitactic mechanisms of motile microorganisms and on gravitropic orientation of higher plants. Consequently, the book Gravitational Biology II—Interaction of Gravity with Cellular Components and Cell Metabolism digs one step deeper into the molecular and physiological basics by discussing the interaction of gravity with biological processes and subcellular structures. In Chap. 1, the authors present evidence that during evolution obviously not only specific sensors for gravity sensing have developed, but that also cells and organisms without specific sensors for the direction of gravity are able to respond to an altered gravitational environment. Here, cell membranes—common to all cells—interact with gravity by changing its fluidity. Single molecules, isolated membranes, and cells have been studied to unravel the basic mechanisms of how gravity affects biological structures and processes. The consequences of these findings for life on Earth in general and specifically for human space travel are addressed. In a hierarchical model from single molecules, neuronal cells via the systems level, to the entire human brain, any impact of altered gravity conditions may lead to serious consequences up to changes in mental and cognitive capabilities of human beings. Changes in membrane fluidity are also known to be relevant for pharmacology. Incorporation of hydrophobic and amphiphilic substances into membranes is clearly dependent on its fluidity. Since many pharmacologically relevant drugs belong to these substances, gravity may lead to changes in drug uptake into cells as could be shown for the model substance alamethicin. At the end of the chapter, the authors present a first functional model of a sensory system based on membrane thermodynamics. Chapter 2 provides detailed insights into the influence of gravity and the consequences of the absence of gravity on gene and protein expression as well as cell metabolism. Again, not the directional information of the gravity vector sensed by organisms via very specific mechanisms and realized in a dedicated and complex signal transduction chain like in gravitaxis and gravitropism is in focus, but the more ix

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general effects on molecular and metabolic processes of cells are described. Mainly based on space experiment data from callus cultures of Arabidopsis thaliana, the present knowledge in the field of gravity-affected cell metabolism is summarized. Especially gravity-induced changes in the flow of calcium ions and of reactive oxygen species (ROS) such as hydrogen peroxide and thus their role as second messengers in metabolic pathways and the importance for cell signaling are discussed. The authors conclude by providing a model for the early signaling events that are initiated by altered gravity stimulation combining the changes in Ca2+ and hydrogen peroxide with fast responses in gene expression, protein modulation, and metabolic pathways. All in all, this book nicely complements the previous publication Gravitational Biology I—Gravity Sensing and Graviorientation in Microorganisms and Plants by providing a view on gravity-induced effects on cellular structures and biological processes—from molecules, cell membranes, and second messengers, via gene and protein expression, to cellular functions. Bonn, Germany August 2018

Günter Ruyters Markus Braun

Acknowledgements

We gratefully acknowledge the financial support by Deutsches Zentrum für Luft- und Raumfahrt (DLR). We are also deeply indebted to PD Dr. Markus Braun for many helpful suggestions in general, as well as to Ulrike Friedrich for her help in parabolic flight campaigns, both from DLR Space Administration. Margret Ecke (University of Tübingen) was a continuously and highly motivated member of the research group from the very beginning. Also in general, we appreciate the support by so many members of the German space industry over the years (ERNO, EADS Astrium, Airbus).

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Contents

1

Interaction of Gravity with Cellular Compounds . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Structure and Function of the Neuronal System . . . . . . . . . . . . . . 1.3 Biological Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Thermodynamics of Neuronal Systems . . . . . . . . . . . . . . . . . . . . 1.6 Interaction of Gravity with Single Molecules . . . . . . . . . . . . . . . 1.7 Interaction of Gravity with Membranes . . . . . . . . . . . . . . . . . . . 1.8 Interaction of Gravity with Neuronal Cells . . . . . . . . . . . . . . . . . 1.9 Membrane Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.10 Action Potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.11 Cytosolic Calcium Concentration . . . . . . . . . . . . . . . . . . . . . . . . 1.12 Discussion and Consequences . . . . . . . . . . . . . . . . . . . . . . . . . . 1.13 Modeling the Gravity Dependence of Neuronal Tissue . . . . . . . . 1.14 Space Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.15 Outlook and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 1 2 4 4 5 6 12 16 16 17 21 22 25 27 29 30

2

Interaction of Gravity with Cell Metabolism . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Opportunities for Exposure . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Plant Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 Determination of Key Metabolites . . . . . . . . . . . . . . . . . . 2.2.4 Metabolic Labeling with (14C)-Glucose . . . . . . . . . . . . . . . 2.2.5 Real-Time Analysis of Ca2+ and Hydrogen Peroxide . . . . . 2.2.6 Gene and Protein Expression . . . . . . . . . . . . . . . . . . . . . . 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 Secondary Messengers: Calcium and Hydrogen Peroxide . . 2.3.3 Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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33 33 35 35 39 39 39 40 40 41 41 44 48 xiii

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2.3.4 Array Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.5 Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.6 Protein Expression and Modulation . . . . . . . . . . . . . . . . . . 2.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

57 60 74 86 87

Chapter 1

Interaction of Gravity with Cellular Compounds

Abstract During evolution, the majority of organisms have developed specific sensors for gravity, the only constant environmental cue on earth. Nevertheless, a variety of gravity effects on molecular, cellular, and physiological level has also been reported in single-cell organisms and cell types of plants and animals which do not seem to possess specific sensors. We have found that the cellular membrane, common to all cells, itself is interacting with gravity by changing its fluidity. Thus, it delivers a basic mechanism for gravity perception for all existing cells and living systems. In the following, we discuss the physical principles and the consequences of our findings for membrane-bound processes, for life on earth, and for manned space travel. In addition, a first model is proposed, how a sensor system for gravity based on membrane thermodynamics could be structured. Keywords Neuronal cells · Gravity perception · Membrane fluidity

1.1

Introduction

Life on earth has evolved under constant 1g gravity conditions, and most organisms have developed specific organs or mechanisms to sense the direction of gravity, to be used, i.e., for orientation in the environment. Such mechanisms exist down even to the level of single cells. However, more general nondirectional gravity effects on molecular, cellular, and physiological processes have been found in many cell types, which are not known to be equipped with specific gravity-sensing mechanisms. This finding gives rise to the question of more basic mechanisms of gravity sensing on the molecular, membrane, or cellular level (Häder et al. 2005). Especially neuronal cells of higher vertebrates are not known to use specific organelles for gravity sensing; however, in case they would be able to sense gravity with other mechanisms, this would possibly have significance for man living in space. To address these questions, experiments have to be performed, studying possible functions of single molecules and cellular membranes in gravity-sensing processes, as there are, for example, the generation and propagation of action potentials and the © Springer Nature Switzerland AG 2018 W. Hanke et al., Gravitational Biology II, SpringerBriefs in Space Life Sciences, https://doi.org/10.1007/978-3-030-00596-2_1

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reaction of chemical synapses in response to changing gravity conditions. In a hierarchical model of neuronal systems, from single molecules to the entire human brain, any gravity dependence in the lower levels would unavoidably lead to serious consequences up to changes in mental and cognitive capabilities of humans in space. Cells including neuronal cells, membranes, and even molecules are highly complex structures and thus have to be looked at within the framework of nonlinear thermodynamics. They can be addressed as either internally or externally energized excitable media giving rise to the consequence that they are critically depending on small external forces to which gravity belongs by definition. This again supports the necessity of experiments with neuronal structures under conditions of changing gravity conditions. Additionally, studying the complex neuronal systems, experiments with simpler model systems, for example, with plain lipid vesicles or oscillating chemical reactions, and computer-based simulations, can be used to explore the gravity dependence of basic biochemical and biophysical processes. Whereas vacuum and radiation conditions as given in space can be simulated on earth within some limits in sufficient quality and duration, platforms to produce microgravity are either of short duration (seconds to minutes) or extremely expensive. The best example is the ISS, by sure the most famous microgravity platform available today. In principle, experiments can be done here at timescales up to years; however, the preparation of the experiments is extremely time-consuming, highly complex, and expensive and the time needed to organize such an experiment can be some years, and the safety regulations are strict. Fortunately, molecular, membrane, and neuronal cellular reactions to gravity changes have been found to be at least partially fast, usually in the millisecond or second to minute range. Thus, mainly short-duration platforms like the drop tower, parabolic flights, and sounding rockets have been conducted in the field of gravitational biology research with membranes, single cells, and action potentials. All microgravity research must be accompanied by proper ground controls and hypergravity experiments. Producing hypergravity is possible with centrifuges even at long timescales and with relatively big experimental setups. If necessary and available, data from ground controls and hypergravity experiments will be presented together with results from microgravity experiments in this chapter.

1.2

Structure and Function of the Neuronal System

Although this chapter mainly focuses on the gravity dependence of molecules, membranes, and cells, a short walk through the systematic structure of central nervous tissue and its connections will help to understand the possible consequences of the gravity dependence in those. The CNS tissue is a highly complex structure, which for our purposes can be seen as a hierarchical system, reaching from single molecules and cells to the entire brain, as depicted in a simplified model in Fig. 1.1. We start from the presence of molecules

1.2 Structure and Function of the Neuronal System

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Fig. 1.1 Hierarchical structure of the human CNS

such as lipids and proteins, which together form the plasma membrane and the membranes of organelles. Membranes create closed biochemical systems, cells, or organelles, which are connected to build tissue and higher structures, as there is finally the entire brain. These cells interact with the environment and among each other by electrical and chemical communication. Action potentials, which are the central electrical communication units in neuronal systems are produced by neurons and propagate along axons to other cells. At the connection between an axon and the next cell, signals are transferred via chemical synapses. Gravity might interact with such a system on all levels of complexity including specific gravity-sensing structures, although it is widely assumed that the human brain itself has no specific gravity-sensing structures. The main sensory input for gravity, position in space, and acceleration conditions to the human brain is the vestibular organ of the inner ear and internal receptors, which are not part of the CNS, and the visual system. Especially the inner ear and the eyes deliver just input channels to the brain. All parts of the human CNS itself are not known to be equipped with any specific gravity-sensing structures. Nevertheless, part of them including single neurons have been shown to react to gravity changes.

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Thus, gravity must interact directly at any level with the system via other mechanisms as will be discussed later in this chapter.

1.3

Biological Membranes

Biological membranes are highly complex systems themselves, mainly composed of lipids, proteins, glycoproteins, lipoproteins, and other molecules. The classical interpretation of membrane function assigns the functional properties mainly to the proteins, whereas the lipids mainly fulfill structural tasks. Proteins can, for example, function as active pumps creating ion gradients by consuming metabolic energy; they can also function as ion channels and as receptors for chemical or physical stimuli. However, even in this simplified view, proteins are always embedded in the lipid matrix of the membrane (Dowhan and Bogdanov 2002). Thus, they will be depending on its thermodynamical properties as there are temperature, fluidity, and other parameters. The cell membrane has, by proper constructs, to interact with other cells and the environment. The communication among cells in neuronal tissue is mainly organized by action potentials propagating along axons and chemical and electrical synapses. In principle, gravity might interact with any of the components of the membrane, mainly proteins, directly. Alternatively, gravity might interact with the membrane as a thermodynamical system or as an excitable medium and thus change its properties. This excitable medium would be energized by ion gradients created by the action of pumps consuming metabolic energy, and the electrical potential (mainly the membrane resting potential) being due to the presence of ion gradients and of selective ion channels, which can be calculated by the Goldman equation (Goldman 1943). In such an interpretation, protein function might be changed by physical membrane properties, especially the membrane fluidity in a secondary step (Aloia and Boggs 1985). Indeed, it has been shown in numerous experiments that the physical state of the membrane is an important factor to modulate protein function. For example, the kinetics of the pore-forming properties of the nicotinic acetylcholine receptor is depending on the physical state of its environment (Zanello et al. 1996).

1.4

Neuronal Cells

Types of cells in the CNS are mainly neurons and glial cells. Neurons are the main signal processing units of the brain, and they are interconnected via dendrites, axons, and chemical synapses. Additionally, electrical synapses are directly connecting cells in the CNS. Action potentials (APs) are generated within a neuron by integration over all input signals and then, via its axon, are delivered to other cells in the CNS. At the

1.5 Thermodynamics of Neuronal Systems

5

interface between axon and the next cell, chemical synapses are the mechanistic units organizing signal transfer. Again, gravity might interact with different structures of such a neuron, even including the intracellular and extracellular matrix. Axons additionally can be seen as cable-like structures in which action potentials propagate. The geometrical and electrical properties of such axons, which are influencing the properties of propagating APs, also might be gravity sensitive. Using the terminology of excitable media, APs are furthermore propagating waves in an excitable medium, which are gravity dependent by definition via the interaction of such media with small external forces.

1.5

Thermodynamics of Neuronal Systems

In short, biological systems including all structures discussed up to now might be seen as excitable media (Epstein and Pojman 1998; Sagués and Epstein 2003; Tabony 2006). Such a medium is based on the following physical needs: • • • • •

It must be thermodynamically open. Mass and/or energy transfer through the system should be present. The system should be far away from equilibrium. Feedback must be present within it. The system must be composed of a large number of small units.

Such a system can be energized internally or externally. In neuronal structures, metabolic energy usually will be the main source. As a consequence of the above mechanism, the system has emerging properties as there are among others: • • • •

Self-organization Pattern formation Oscillations Propagating waves

and the system in its properties is critically dependent on small external forces including gravity as a small physical force. Of course, an excitable medium has to obey the first and second law of thermodynamics. Usually excitable media are described in terms of nonlinear thermodynamics. The mathematical presentation is given by coupled sets of nonlinear differential equations or possibly for computer-aided simulation by cellular automata (Acedo 2009; Wolfram 2002) or other formalistic systems. Applying the just stated theoretical approaches to neuronal systems, it becomes obvious that the brain, parts of it, but also single cells can be interpreted as excitable media (Wiedemann and Hanke 2002); however, plain membranes or vesicles with identical internal and extracellular aqueous solution are not excitable media, as they are energetically close to equilibrium systems with only thermodynamical fluctuations. Another important fact about neuronal systems (biological systems in general) is that the biological membranes of neurons may be described as two-dimensional

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Fig. 1.2 Monolayer on a film balance. The film pressure is plotted as function of the area per lipid molecule. The plateau is due to the main phase transition of the lipid

systems, which are defined by temperature, lateral pressure, and area. Accordingly, at constant temperature as usually given in neuronal systems, such a membrane can be described by a graph plotting lateral pressure against area. To do so for a real bilayer mechanistically is very difficult, but using a lipid monolayer on a film balance as a model system, such curves can be measured easily as shown in a simplified example in Fig. 1.2. Here, a graph showing the film pressure as a function of area is given for a plain lipid with a defined phase transition temperature, as can be seen in the graph. According to the Singer-Nicolson fluid mosaic model (Singer and Nicholson 1972), biological membranes are usually in the fluid state but might be shifted along the curve describing its state. As biological membranes are of highly complex structure, composed of different lipids, proteins, and other molecules, the situation usually will not be as simple as given in Fig. 1.3. However, even in real membranes, phase transitions might occur, and any membrane and more specific protein function will depend on the actual position of the system membrane in the physical space.

1.6

Interaction of Gravity with Single Molecules

A very basic question in the field of gravity research is whether gravity directly changes the properties of single molecules with relevant biological function. One of the main functional units of neuronal membranes is ion channels formed by different proteins. They are involved among others in ion homeostasis, signal transduction, and the generation of action potentials. Typical structural elements of such ion channels are alpha-helical and beta-sheet parts of the molecule spanning the membrane. Typical properties of an ion channel are the opening and closing times and the

1.6 Interaction of Gravity with Single Molecules

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Fig. 1.3 The sketch in the upper part shows the principals of a bilayer experiment; below, a photo of a setup for bilayer experiments used in parabolic flight missions is shown [modified from Wiedemann et al. (2011)]

open-state probability and its selectivity. Additionally, it must be considered by which parameters the ion channel is controlled, usually by ligands or by membrane potential. Technically, the abovementioned parameters of ion channels can be investigated best by electrophysiological techniques like the patch-clamp technique (Hamill et al. 1981) or the reconstitution of proteins into planar lipid bilayers (Hanke and Schlue 1993). With such techniques either single-channel studies or voltage- or current-clamp experiments can be done. In Fig. 1.3, the basics of a bilayer experiment are shown together with a photo of a setup used in parabolic flight experiments. In Fig. 1.4, the patch-clamp technology is depicted, and the technology used for patch-clamp experiments under microgravity in a parabolic flight mission is shown (Meissner and Hanke 2002). Experiments utilizing both techniques were done in parabolic flight campaigns and in the drop tower. According to the mechanical sensitivity of patch-clamp experiments and the need for sensitive handling by the scientist in parabolic flight campaigns, only a few

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Pipette solution Pipette Seal resistance 1 – 100 GΩ

Pipette diameter 1–5 μ m Membrane

Ion channel

Cytoplasma

Fig. 1.4 The upper part shows the basics of the patch-clamp technology. The photo in the lower part shows an older setup as used in parabolic flight missions. This setup also has been adapted to the drop tower [modified from Wiedemann et al. (2011)]

such experiments were performed, and data are still rare. With the development of the planar patch-clamp technology, semiautomatic equipment for patch-clamp experiments became available. Due to the much easier handling and to advances in mechanical stability, later, mainly this technique was used in microgravity experiments in parabolic flight missions (i.e., Wiedemann et al. 2011). The basics of this technology are depicted in Fig. 1.5, and a photo of a real setup is shown in Fig. 1.6. Here a commercial setup, the Port-a-Patch system from Nanion®, was adapted for the experiments. Regardless of the advances in technology, single native ion-channel electrophysiological experiments have been found to be extremely difficult to conduct under microgravity conditions (Kohn 2010); thus, published results are rare. Some experiments with the model pore alamethicin and with porins, however, have delivered useful data in the field. The alamethicin pore is created by bundles of alpha helices in a barrel-like fashion (Boheim 1974; Hall et al. 1984; Woolley and Wallace 1992;

1.6 Interaction of Gravity with Single Molecules

9

Fig. 1.5 This sketch compares the classical patch-clamp technique to the planar patch-clamp technology tower [modified from Wiedemann et al. (2011)]

Leitgeb et al. 2007) and is a well-suited and deeply studied model system for ion channel research (Boheim et al. 1983, 1984; Cafiso 1994). In direct single-channel studies in planar lipid bilayers, it was found that the activity of alamethicin-induced pores is gravity dependent (Hanke 1995; Klinke et al. 2000; Wiedemann et al. 2003). The overall pore activity increases via increasing gravity. The same was found for a pore-forming porin reconstituted into a planar lipid bilayer (Wiedemann et al. 2003, 2011). An example is shown in Fig. 1.7. Single alamethicin data are shown in Fig. 1.8. It should be mentioned here, however, that optical techniques are increasingly been used in the field and might be the future of investigating functional properties of ion channels. Such new optical techniques possibly in some time can be used in the field of “live sciences under space conditions” and will help to obtain the required data. In addition, in experiments using oocytes with overexpressed epithelial sodium channels, evidence was found that these channels close toward reduced microgravity and open upon hypergravity (Richard et al. 2012). The experiments were performed in a parabolic flight mission. Measurements of whole-cell currents utilizing the patch-clamp technique resulted in a more complex dependence of membrane permeability on gravity (Fig.1.9). This is most probably given by the fact that wholecell currents usually are a complex mixture originating from a variety of active channels and transporters. In laboratory experiments, usually pharmacological interventions are done, for example, to block part of the membrane conductance of the cell by proper drugs (Wiedemann et al. 2011). The use of most of the relevant drugs to block certain ion-channel populations or transporters (i.e., to block sodium channels by TTX), unfortunately, is not allowed in parabolic flights, as usually they are significantly toxic. This violates safety regulations given for parabolic flights (Novespace 2009) and air travel in general.

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Fig. 1.6 Photo of a semiautomated patch-clamp setup for parabolic flight missions utilizing the planar patch technique with the Port-a-Patch system from Nanion®. Below, details of the experimental box are shown tower [modified from Wiedemann et al. (2011)]

Another approach to measure currents across cell membranes under variable gravity conditions was chosen by Richard et al. (2012). They recorded currents across oocyte membranes in a parabolic flight experiment with a specifically designed setup, and they found a significant reduction of current under microgravity. This could be interpreted as closing of ion channels in the membranes of the oocytes and thus would be consistent with our results. Additionally, from a variety of experiments in so-called simulated microgravity, using clinostats or random positioning machines, but also mechanical unloading, data has been published suggesting a reduced ion-channel activity under microgravity. According to our statement about the physical questions behind these technologies, we will not comment the results in detail here but just point out that, when being used, they support the above given results from real microgravity experiments.

1.6 Interaction of Gravity with Single Molecules

11

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4 3 2 1 0

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Fig. 1.7 Dependence of single porin pore fluctuations on gravity. Traces of alamethicin fluctuations are shown at different gravity levels [modified from Goldermann and Hanke (2001)]

Fig. 1.8 The open-state probability of alamethicin pore fluctuations on gravity is shown. Obviously, pore activity increases toward higher gravity levels (Wiedemann et al. 2011)

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Fig. 1.9 Current voltage relation of a whole cell under conditions of variable gravity (Wiedemann et al. 2011)

1.7

Interaction of Gravity with Membranes

Biological membranes, as stated already, are complex constructs mainly from proteins and lipids. Aside from the functional properties as implemented by the proteins, they have some gross thermodynamical properties of interest. One of the major points of interest is the question in which physical state, fluid or crystalline, the membrane is, having however in mind that due to the structure of membranes, as basically described by the fluid mosaic model (Singer and Nicholson 1972), they are usually in the fluid state. The fluidity of membranes can best be investigated using fluorescent dyes to measure fluorescence polarization anisotropy (FPA) (Lacowicz 2006) in real cell membranes but also in plain lipid membranes and membranes of liposomes (Fig. 1.10). In our studies, we have used human neuroblastoma cells in the original and in the re-differentiated state. Alternatively, pure lipid membranes, as given by artificial vesicles, have been used. These deliver a simpler model of a cell membrane allowing concentrating on the biophysical basics (Torchilin and Weissig 2003). Another difference between cells and liposomes is the presence of the cytoskeleton in cells, which interacts with the cell membrane and most probably modifies its response to gravity changes. Technically, the FPA measurements were done in a commercial multimodal, multi-plate reader prepared for parabolic flights shown in Fig. 1.11. The results for the vesicle measurements are given in Fig. 1.12, those for the cells in Fig. 1.13. In both sets of experiments (Sieber et al. 2014), vesicles and real cells, membrane fluidity increases under microgravity. An exception is given, when liposomes at a temperature in the range of the phase transition temperature of the lipid are used. Here, no fluidity changes due to gravity changes were found. This is due to the fact that membranes are highly compressible in this state. In an additional set of experiments, the size of plain lipid vesicles was investigated in the drop tower using the light-scattering technology. In these experiments, it was found that the vesicles became slightly bigger under microgravity

1.7 Interaction of Gravity with Membranes

13

Fig. 1.10 Technology of the fluorescence polarization anisotropy measurements using DPH as fluorescent dye [modified according to Zhao and Lappalainen (2012)]

Fig. 1.11 A setup build around a 96-well universal plate reader for parabolic flight missions is shown

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Fig. 1.12 Fluidity dependence of membrane fluidity in plain lipid vesicles. Increasing FP depicts decreasing fluidity. In the fluid state of the membrane, fluidity increases toward microgravity, left part. In the phase transition range of a given lipid, lower part, no gravity dependence of fluidity was found [modified from Sieber et al. (2014)]

(Wiedemann et al. 2011). In experiments with real cells (insect embryonic cancer cells), no changes in cell size and geometry were detected within the experimentally given resolution. This might be due to the fact that the cytoskeleton of the cells is preventing such changes. In a given lipid membrane, membrane fluidity is correlated to the lateral pressure of the membrane (which is directly related to the membrane fluidity). In case the lateral membrane pressure changes, due to the correlation between pressure and area in an open system, the area of the membrane also changes (see Fig. 1.2). By this, the size of the vesicles might change due to gravity changes. Further experiments with plain lipid bilayers were done utilizing the planar lipid bilayer voltage-clamp technique at high membrane potential (V > 100 mV). Under

1.7 Interaction of Gravity with Membranes

15

Fig. 1.13 Gravity dependence of membrane fluidity of neuronal cells. In both cell versions used, membrane fluidity increases with decreasing gravity levels [modified from Sieber et al. (2014)]

Fig. 1.14 Current fluctuation density at high membrane potential in asolectin bilayers at different gravity levels. Current fluctuation density induced by high potentials is reduced at microgravity [modified from Sieber et al. (2016)]

these conditions, current fluctuations can be induced in the plain lipid bilayer, which might be interpreted, for example, as lipid ion channels (i.e., (Heimburg 2010)). Such current fluctuations can be measured; the integral current density fluctuation in a bilayer measured in a parabolic flight mission is shown in Fig. 1.14. It decreases toward lower gravity levels.

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1.8

Interaction of Gravity with Cellular Compounds

Interaction of Gravity with Neuronal Cells

As stated above, gravity might interact directly with cells having no explicit structure for gravity sensing, and, indeed, it has been shown that a variety of such cells including neurons respond to gravity changes. Possible changes to be expected (and having partially been found) in such cells are: • • • • • • •

Structural changes Changes in shape Changes in composition Changes in the cytoskeleton Changes of the electrical properties Gene expression changes Immune response changes

In addition, there are some more general differences relevant for cells to be found under microgravity conditions compared to 1g: • • • • • • •

No convection. No sedimentation. Access to nutrients becomes diffusion limited. Waste dissipation becomes diffusion limited. Decreased hydrodynamic shear. This in some examples has been shown to effect. Membrane fluidity (Mallipattu et al. 2002).

Neurons are typically cells without specific gravity-sensitive structure and de facto also without any known need for it. Especially their electrical properties are defining the signal processing in the brain, and, accordingly, the clearly proven gravity dependence of electrical properties of neuron will be discussed here in more detail. This is also due to the finding, see above, that ion-channel properties are strictly gravity dependent, and ion channels are significantly involved in the electrical properties of cells.

1.9

Membrane Potential

One basic property of any cell is its resting membrane potential defined by the ion gradients and the selective membrane permeability given by ion channels, especially potassium channels. The ion gradients are established mainly by pumps consuming metabolic energy (ATP). The Goldman equation (or in simplified situations the Nernst equation) can be used to calculate the membrane potential. This membrane potential usually is measured by electrophysiological techniques utilizing, i.e., glass microelectrodes with electrometer amplifiers. Meanwhile, a variety of

1.10

Action Potentials

17

Fig. 1.15 Membrane potential changes in spherical SF 21 insect cells measured with the dye D-4ANNEPS. The membrane potential becomes slightly more positive, indicating a depolarization of the cell membranes [modified from Wiedemann et al. (2011)]

potential-dependent fluorescent dyes is available, and optical techniques have entered the field of membrane potential measurement. In a variety of different cells, membrane potential changes as a consequence of altered gravity have been reported (i.e., Wiedemann et al. 2011). As an example, in Fig. 1.15, the response of the membrane potential of an embryonic insect cancer cell to a gravity change is shown, as has been measured in an optical drop-tower experiment. A small membrane depolarization of an embryonic insect cancer was found as a consequence of the exposure of the cell to microgravity measured by the fluorescent potential-sensitive dye D-4-ANNEPS. Membrane potential changes due to gravity changes have also been published for other unicellular organisms including neuron-like cells (Kohn 2013). These results were not completely systematic; sometimes, a hyperpolarization due to microgravity exposure was mentioned. In any case, in a neuron, changes in the membrane resting potential will result among others in differences in action potential initiation and propagation.

1.10

Action Potentials

Neurons in neuronal tissue process all incoming signals by integration, which leads to a specific membrane potential. In case the membrane is sufficiently depolarized relative to the resting potential of the cell, an action potential (AP) is generated in the cell. Neurons communicate by such action potentials, which are propagating from

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the firing cell to the next one along an axon, and they are passed to the next cell via chemical synapses. One main parameter classifying an action potential is its propagation velocity, which is defined by the geometrical and electrical properties of the axon as given in the following equation for the simpler case of a none myelinated axon (Tasaki 2004): sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi d v¼ 8  ρ  C 2  R∗ where C is the membrane capacity, R is the membrane resistance, d is the diameter of the axon, and p is the resistivity of the axoplasm. The propagation velocity of an AP among others is critically depending on the properties of the involved ion channels defining the membrane resistance and on the geometry of the axon. In case, for example, the open-state probability of voltagedependent ion channels is changed by gravity changes, see above, this should directly result in changes of the propagation velocity of the AP. Of course, especially voltage-gated sodium channels are relevant here, but for these up to now, no singlechannel data under microgravity is available. However, when assigning the fact from above that some ion channels close upon microgravity to the axon membrane ion channels, the R in the equation would become bigger and the velocity would slow down, just consistent with our data. Additionally, in case a membrane becomes more fluid, it becomes thinner due to the higher mobility of the fatty acid chains of the lipids; thus, the capacity of the membrane becomes higher as it is C ¼ ε  ε0  A=d where C is the capacity, ε the dielectrical constant of the medium (about 2 for lipids), ε0 the general dielectrical constant, A the area, and d the thickness of the membrane. In addition, the axonal conductivity in case of a massive ion influx would increase, again in consistence with the other data. Experiments concerning the question of action potential propagation velocity under microgravity using muscle APs in EMG and related studies have been done already as early as in the ninetieth of the last century (Layne and Spooner 1990) and in the following years (Rüegg et al. 2000). These experiments among others clearly showed the gravity dependence of muscle action potential propagation velocity (Pandis et al. 2009). Also, in plants using Dionaea muscipula (Pandolfi et al. 2014), parabolic flight experiments were performed to investigate whether the electrical signaling of trap closure of this plant is depending on gravity, and it was suggested from the results that possibly the generation or propagation of the plant action potential was directly depending on gravity. More detailed experiments were then done with isolated axons from rat (myelinated axon) and intact earthworm (good model system for none-myelinated axons), which demonstrated a basic dependence of the propagation velocity of action

1.10

Action Potentials

19

Fig. 1.16 In the experiment shown, an earthworm (or alternatively the rat ischiadicus) was prepared and placed in a proper chamber, here shown for the earthworm experiment. To this chamber, measuring electrodes (Ag/AgCl) were connected as shown in the sketch. Current pulses inducing membrane depolarization and thus initiating action potentials were applied to the fiber/ ischiadicus. In the recording given at the lower left side from an earthworm, the stimulus artifact can be seen in both channels, followed by the recording of the propagating action potential. The propagation velocity was calculated from the time difference at the second recording electrode sets and the distance between these electrodes. Figure modified from Meissner and Hanke (2005)

potentials on gravity. A sketch of a typical experimental setup for a parabolic flight experiments with earthworm is shown in Fig. 1.16, together with an experimental trace. By such experiments, it was clearly shown that APs propagate slower in microgravity (Wiedemann et al. 2011). The second question related to the gravity dependence of AP properties is in how far their initiation is modified. As shown above, the membrane resting potential is gravity dependent. The relative value of the membrane potential compared to the threshold for action potential initiation is defining how easy an action potential can be triggered. To answer the question whether AP onset is gravity dependent,

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Fig. 1.17 Recording from a spontaneously firing neuron from leech in a drop-tower experiment. The overall potential recording from an intracellular electrode is given together with the g-value. At 2.2 s, the g-sensor indicates the release of the capsule. The artifact at about 7 s results from the arrival of the setup at the breaking unit of the drop tower. The two insets show the potential recordings at higher time and amplitude resolution at 1g (left) and microgravity (right). The number of action potentials increases under microgravity. Figure modified from Meissner and Hanke (2005)

spontaneous spiking leech neurons were used in a drop-tower experiment, and it was shown that the number of spikes increases under microgravity as shown in Fig. 1.17. A first basic explanation of the presented results can be deduced from the results presented for the resting potential of cell membranes above. A small depolarization will lower the level for the initiation of an AP, and thus in the given experiment, the number of spontaneous APs will become bigger. Related to the question of AP onset is the latency, the period between a proper stimulus and the onset of the AP. The shorter the latency is the easier is an AP to elicit. In the simplest case, this is analogue to the amount of membrane depolarization. Unfortunately, the terminus latency is often used more generally in physiological experiments, for example, describing the response of a muscle to an external stimulus. In such cases, not only the before defined latency but also AP propagation velocity and the behaving of the involved synapses is involved. Thus, it is difficult to compare the results in the literature. It is important to mention here that there is no conflict in the statements that first, action potentials propagate slower on axons in microgravity and, second, are easier to elicit. In the first case, we are talking about a propagating wave; in the second case, an equipotential situation in a cell is given without propagation. Due to the idea to look at membranes and cells as excitable media, it is of interest to have a look at other systems, which are discussed under the same assumption. Such systems can be used as model systems in some cases, as they possibly easier to be used in microgravity experiments or deliver additional information of interest.

1.11

Cytosolic Calcium Concentration

21

Two of these systems are of more specific interest, as to both the Hodgkin-Huxley formalism of action potentials has been adapted. The Belousov-Zhabotinsky reaction (Belousov 1959; Zaikin and Zhabotinsky 1970) is a chemical reaction exhibiting oscillatory behavior as well as propagating waves in thin layers of fluid or gels. BZ waves propagate much slower (5 mm/min) than APs and their mechanism is completely different. They are reaction-diffusion waves based on coupled chemical reactions. Nevertheless, propagating waves in the BZ can be described using similar differential equations as being used to describe propagating action potentials (i.e., Murray 2002) or any other propagating wave in an excitable medium. They also show the same behavior as action potentials in some other aspects; they annihilate upon collision, and they have a defined absolute and relative refractory period. Finally, their propagation follows a nonlinear dispersion relation as usually found for waves propagating in excitable media. Indeed, it was found that the propagation velocity of BZ waves depends on gravity too, but they speed up under lower gravity. The other system having some similarities to action potentials but also to the BZ reaction is the retinal spreading depression (Fernades de Lima et al. 2015; MartinsFerreira and de Oliveira-Castro 1966), a reaction-diffusion wave traveling through retinal tissue with a speed of about 5 mm/min, thus, similar to the BZ reaction. Again, its speed and its initiation are gravity dependent but much more complicated than found for action potentials. This is most probably due to the fact that here we are looking at a wave in neuronal tissue with its highly complex structure.

1.11

Cytosolic Calcium Concentration

As has been shown above already, the membrane resting potential of cells in most cases under microgravity slightly depolarizes. Most cells including neurons have potential-sensitive calcium channels in their membranes, which open upon depolarization. These channels might change their open-state probability under microgravity. Usually a high calcium gradient exists over cell membranes. Taking these facts into account, changes in intracellular calcium concentration, possibly an increase of intracellular calcium concentration, can be expected under microgravity. Accordingly, experiments have been done in parabolic flight using neuronal cells together labeled with the dye Fluo-3 AM, which is sensitive to the intracellular calcium concentration. The experimental setup had been built around a commercial 96-well multipurpose plate reader as previously shown already. The result of such a measurement is shown in Fig. 1.18 (Hauslage et al. 2016). Similar results were found in clinorotation experiments using the same cells (Hauslage et al. 2016) and also in Arabidopsis thaliana cells (Hausmann et al. 2014). Opening of membrane potential-dependent calcium channels due to membrane depolarization easily can explain these findings. Interestingly, in the experiments by Hauslage et al. but also in Arabidopsis thaliana cells, it was found that under hypergravity the intracellular calcium

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Fig. 1.18 Relative fluorescence of Fluo-3 AM incubated SH-SY5Y cells during parabolic flights. Intracellular calcium concentration increases significantly with decreasing gravity levels [modified from Hauslage et al. (2016)]

concentration decreases (Neef et al. 2015). This indicates that under hypergravity conditions calcium pumps must be activated further decreasing the intracellular calcium concentration. This would require a signal transduction cascade in the cells to be triggered by hypergravity activating ATP-driven or secondary energized calcium pumps.

1.12

Discussion and Consequences

The most general finding within the above presented results is the fact that the fluidity even of plain lipid membranes is gravity dependent (see also Klymchuk et al. 2006; Kordyum et al. 2015). The fluidity increases toward lower gravity values. To explain this finding mechanistically is not trivial. Our statement that biological systems behave like excitable media is no valid here, as plain liposomes with identical intra- and extraliposomal aqueous solution are not far enough from equilibrium. Thus, the fluidity change cannot be energized by electrochemical gradients or metabolic energy. The most reasonable explanation to understand the effect is that it is thermodynamical driven. This should result in a small temperature shift of the system to lower values, which however, due to the high buffer capacity of the used setup, cannot be measured. Thus, to create an experiment, tackling this question will be of high biophysical importance. Alternatively, taking into account that mot membranes are not flat, but have certain ripple structures, interaction of gravity with this more complicated threedimensional structure might be relevant. Coming now to the consequences of membrane fluidity being gravity dependent, it is obvious that by this any cell will have a residual most probably small gravity dependence. This has been due of course from the first cell on earth to today, too,

1.12

Discussion and Consequences

23

Fig. 1.19 Dependence of nicotinic AChR channel parameters on membrane fluidity as measured by fluorescence. The ion channel conductance increases toward higher fluidity, left side, and the closed-state probability of the channel decreases toward higher fluidity, this being in agreement with our findings for model pores polarization [modified from Zanello et al. (1996)]

and, thus, evolution has taken place under the permanent recognition of a stable gravity of about 9.8 m/s2 (1g). Accordingly, some aspects of evolution should possibly be discussed taking this into account. In higher organisms, having specific gravity sensors, the effect of membrane fluidity being gravity dependent might be ignorable, but not in single cells without such sensors as, for example, in neurons, but also in single-cell protists like Euglena gracilis. Especially according to the effect on neurons, a basic dependence of mental (brain) functions on gravity can no longer be excluded. Indeed, a variety of findings have been presented toward this direction (i.e., Wiedemann et al. 2011). However, the data about effects of gravity on brain function are complex, partially confusing, and even sometimes contradictory. In addition, they depend a lot on the set of the specific experiment. Thus, we will not go into details about gravity effects on the CNS here but will focus on membrane and cellular aspects. It is furthermore accepted meanwhile that the function of membrane intrinsic proteins among others is depending on thermodynamical membrane parameters. Among these membrane parameters by sure is the membrane fluidity, and it has, for example, been shown that the parameters of the ion channel of the nicotinic acetylcholine receptor (nAChR) are directly depending on membrane fluidity as presented in the following figure from the literature. From the results of Fig. 1.19, it can be concluded that the open-state probability of the nicotinic AChR decreases toward higher fluidity and thus toward lower gravity, which is consistent with our above presented results for the model pore alamethicin and the porin pore. The finding that the open-state probability of ion channels is gravity dependent, thus, just is a consequence of the change of membrane fluidity. Among others, the complete chemical synaptic transmission in neuronal systems and in the periphery should be gravity dependent.

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Fig. 1.20 Increasing the lateral pressure in a bilayer (arrow) in a double filmbalance setup increases the activity of incorporated alamethicin [modified from Hanke and Schlue (1993)]

Furthermore, as the behavior of receptor proteins is membrane fluidity dependent, see the nAChR results shown above, the binding constants of ligands to other membrane receptors should also be gravity dependent. A major part of all relevant pharmacological drugs being used presently belongs to the substances binding to G-protein-coupled receptors (7-transmembrane-alpha-helix receptors, i.e., the muscarinic AChR). Accordingly, the activity of most of the relevant pharmacological substances might be gravity dependent. This must be considered seriously at least in later longer-lasting manned space missions. Finally, as stated already, the function of any other membrane protein could become gravity dependent, as, for example, ABC membrane transporters (Vaquer et al. 2014) or thyrotropin receptors (Albi et al. 2011). Using alamethicin as sensor for lateral membrane pressure, it has been shown earlier (Hanke and Schlue 1993) that upon increasing the lateral pressure in a bilayer using a double film-balance setup, the activity of alamethicin also increases. This is consistent with higher activity at lower fluidity and at higher gravity values (Fig. 1.20). Generalizing the finding that ion channels tend to close toward microgravity and using the equation for action potential as given above allows to state that the membrane resistance of axons will become higher and ,thus, action potentials will slow down under microgravity, as indeed has been shown in our experiments. Additionally, it was shown that the frequency of spontaneous action potentials in isolated neuron increases under microgravity, a finding being just the result of the depolarization of cells under microgravity. This depolarization in our interpretation comes from the closing of potassium channels under microgravity, which leads to a membrane resting potential depolarization as can be related from the Goldman equation. For this statement, we just have to use the additional dogma that the resting potential of cells almost exclusively is determined by the selective permeability of the membrane for potassium (as can be calculated by the Nernst equation). It is important, however, to point out that spontaneous action potentials in isolated spiking neurons are not propagating, and, thus, their behavior is not directly related to the propagation of action potentials along axons.

1.13

Modeling the Gravity Dependence of Neuronal Tissue

25

Fig. 1.21 Scheme of a sensory system

1.13

Modeling the Gravity Dependence of Neuronal Tissue

In sensory physiology, it is typically assumed that a proper sensor protein is given for the sensory stimulus in the plasma membrane of a given sensory cell. The sensory cell is embedded in a complete hierarchic system as shown in Fig. 1.21. This receptor is located in the sensory cell membrane and might be an ion channel, directly activated by a stimulus, as, for example, pH-sensitive channels for acid recognition in the case of taste. It can also be a classical 7-transmembrane-helix receptor as given for olfactory stimuli or in vertebrate vision. Either such a receptor induces a direct membrane potential change or activates a second messenger cascade inducing again membrane potential changes but possibly also other actions. Let us assume that it is allowed to make the membrane fluidity the sensory receptor for gravity, a physical property thus being a receptor for a physical stimulus. Then, the sensory transduction cascade can be redesigned in relation to classical sensory cascades as given in Fig. 1.22. With these assumptions, we can now design a model from the above findings starting with microgravity as a stimulus being sensed by the receptor ”membrane fluidity” and go forth to its final influence on coupled neurons as given in Fig. 1.23.

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Fig. 1.22 Comparison of a classical sensory cascade, right side, with a restructured model of gravity perception, left side. Aside from the effect of the input signal to ion channels as given here as an example, second messenger cascades as well as membrane fluidity can induce many other processes in cells as depicted in the scheme [modified from Kohn et al. (2017)]

Fig. 1.23 The interaction of gravity with neuronal systems, from membrane fluidity to the coupling of neurons [modified from Kohn and Ritzmann (2017)]

1.14

Space Pharmacology

27

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Fig. 1.24 Dependence of alamethicin incorporation into monolayers of different starting pressure [modified from Volinsky et al. (2004)]

1.14

Space Pharmacology

There is another major consequence of membrane fluidity changes due to gravity changes, as already shortly mentioned, related to later manned space missions, which is related to the relevance of the above shown findings to pharmacology. It is well known that the incorporation of hydrophobic and amphiphilic substances into membranes is clearly depending on lateral membrane pressure and thus on fluidity. A significant number of relevant pharmacologically relevant drugs including, for example, anesthetics, steroids, and others, belong to this group of substances. An example from the literature, again using alamethicin as sensor, is shown in Fig. 1.24. Here, in monolayers of different starting pressure, alamethicin was incorporated with clearly pressure-dependent kinetics (Volinsky et al. 2004). From Fig. 1.24, a problem with the timescale becomes obvious. Usually the incorporation processes of hydrophobic and amphiphilic substances into membranes are relatively slow; thus, parabolic flight or other short-time microgravity platforms are not suitable for related experiments. Unfortunately, presently, no long timescale experiments concerning this question are available, but at least the data from one parabolic flight mission demonstrated that the kinetics of the incorporation of alamethicin into vesicle membranes is gravity dependent. Thus, it was tested whether the incorporation of hydrophobic substances into membranes might be gravity dependent, as it was known that the incorporation of substances into membranes is depending on membrane fluidity (see also later in the text).

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Fig. 1.25 Incorporation kinetics of alamethicin into vesicle membranes. Alamethicin was added to the vesicles at the beginning of each gravity phase in a modified stop-flow apparatus; this could be seen by the artifact at the beginning of each trace. The upper part shows the recording of a complete parabola, below, at an extended timescale is given for the 1g phase and for the microgravity phase. As can be seen, the slope of the incorporation is different

As a model system, plain lipid vesicles were used, to which alamethicin was added. Changes in lipid fluidity as measured by fluorescence polarization anisotropy were taken as a measure for alamethicin incorporation. The result is shown in Figs. 1.25 and 1.26. The fluorescence polarization anisotropy technique was used for the experiments (data not yet published), and it was found that the incorporation kinetics of alamethicin into the vesicle membranes is changed under microgravity, being possibly a bit faster here.

Outlook and Future Perspectives

Fig. 1.26 Data evaluation from traces as shown in the previous figure. The incorporation of alamethicin into membranes is gravity dependent probably being slightly increased under microgravity; the results for the 1.8 g phases are scattering; thus, they are not discussed here

relative incorporation rate alamethicin (slope)

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1.15

Outlook and Future Perspectives

Although a lot of information has been acquired about the effects of gravity on living systems, there is still a significant lack of important data. Systematic data about single-channel behavior under microgravity is still missing. Electrophysiological experiments under microgravity conditions up to now have been shown to be difficult, but possibly. Other techniques, mainly optical approaches, might deliver new possibilities for such measurements. It is also obvious that the timescale of a variety of experiments is a problem together with the available microgravity platforms. In the above presented story, this is especially due for pharmacological experiments but also for many other questions. What is needed is microgravity in the range of one up to a few hours. The short-time platforms like sounding rockets cannot deliver much more than about 15 min of microgravity. By using orbital platforms, it would be technically feasible, but would be far too expensive, when only hours are needed. New approaches to solve this problem by sure would be welcome within the scientific community. In case physiological functions are reacting to microgravity, it is obvious that they also should be reacting to hypergravity. Consequently, more experiments under high gravity should be done to expand our current knowledge. Such experiments can be done on centrifuges without relevant restrictions in time and in acceleration (gravity) level. Finally, the problem of so-called simulated microgravity should be reconsidered, and it should be clarified to which extent and for which questions these techniques can be used. According to the presently available information in the literature, mainly clinostats might be useful here to some extent.

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Interaction of Gravity with Cellular Compounds

References Acedo LA (2009) Cellular automation model for collective neural dynamics. Math Comput Model 50:717–725 Albi E, Ambesi-Impiombato FS, Peverini MM, Damaskopolus E, Fontanini E, Lazzarini R, Curcio F, Perella G (2011) Thyrotropin receptor a membrane interactions in FRTL-5 thyroid cell strain in microgravity. Astrobiology 11:57–64 Aloia RC, Boggs JM (1985) Membrane fluidity in biology. Academic Press, Orlando, FL Belousov BP (1959) Eine periodische Reaktion und ihr Mechanismus (translated from Russian to German). In Sbornik referatov po radiacionoj medicine za. Moskau 147:145 Boheim G (1974) Statistical analysis of alamethicin channels in black lipid membranes. J Membr Biol 19(1):277–303 Boheim G, Hanke W, Jung G (1983) Alamethicin pore formation, voltage dependent flip-flop of a-helix dipoles. Biophys Struct Mech 9:188–197 Boheim G, Hanke W, Jung G (1984) The alamethicin pore is formed by a voltage-gated flip-flop of a-helix dipoles. In: Welter W et al (eds) Chemistry of peptides. W. de Gruyter, Berlin, pp 281–289 Cafiso DS (1994) Alamethicin: a peptide model for voltage gating and protein-membrane interactions. Annu Rev Biophys Biomol Struct 23:141–165 Dowhan W, Bogdanov M (2002) Functional roles of lipids in membranes. In: Vance DE, Vance JE (eds) Biochemistry of lipids, lipoproteins and membranes. Elsevier, Amsterdam, pp 1–35 Epstein IR, Pojman JA (1998) An introduction to nonlinear chemical dynamics. Oxford University Press, Oxford. ISBN 978-0195096705 Fernades de Lima VM, Piqueira JRC, Hanke W (2015) The tight coupling and non-linear relationship between the macroscopic electrical and optical concomitants of electrochemical CNS waves reflect the non-linear dynamics of neural glial propagation. Open J Biophys 5(1):11 Goldermann M, Hanke W (2001) Ion channels are sensitive to gravity changes. J Microgravity Sci Technol XIII/1:35–38 Goldman DE (1943) Potential, impedance, and rectification in membranes. J Gen Physiol 27:37–60 Häder D-P, Hemmersbach R, Lebert M (2005) Gravity and the behavior of unicellular organisms. Cambridge University Press, New York, NY Hall JE, Vodyanoy I, Balasubramanian TM, Marshall GR (1984) Alamethicin. A rich model for channel behavior. Biophys J 45(1):233–247 Hamill OP, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for highresolution current recordings from cells and cell-free membrane patches. Pflügers Archiv 391:85–100 Hanke W (1995) Studies of the interaction of gravity with biological membranes using alamethicin doped planar lipid bilayers as a model system. Adv Space Res 6/7:143–150 Hanke W, Schlue W-R (1993) Planar lipid bilayer experiments: techniques and application. Academic Press, Oxford Hauslage J, Albrecht M, Hanke L, Hemmersbach R, Koch C, Hanke W, Kohn PM (2016) Cytosolic calcium concentration changes in neuronal cells under clinorotation and in parabolic flight missions. Microgravity Sci Technol 28(6):633–638. https://doi.org/10.1007/s12217-016-9520-y Hausmann N, Fengler S, Henning A, Franz-Wachtel M, Hampp R, Neef M (2014) Cytosolic calcium, hydrogen peroxide and related gene expression and protein modulation in Arabidopsis thaliana cell cultures respond immediately to altered gravitation: parabolic flight data. Plant Biol 16(Suppl 1):120–128 Heimburg T (2010) Lipid ion channels. Biophys Chem 150:2–22 Klinke N, Goldermann M, Hanke W (2000) The properties of alamethicin incorporated into planar lipid bilayers under the influence of microgravity. Acta Astron 47:771–773 Klymchuk DO, Baranenko VV, Vorobyova TV, Dubovoy VD (2006) Fluidity of pea root plasma membranes under altered gravity. http://adsabs.harvard.edu/abs/2004cosp.35.1356K

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Kohn F (2010) Patch clamp experiments with human neuron-like cells under different gravity conditions. PhD thesis. University of Hohenheim, Stuttgart, Germany Kohn F (2013) High throughput fluorescent screening of membrane potential and intracellular calcium concentration under variable gravity conditions. Microgravity Sci Technol 25(2):113–120 Kohn FPM, Ritzmann R (2017) Gravity and neuronal adaptation, in vitro and in vivo-from neuronal cells up to neuromuscular responses: a first model. Eur Biophys J. https://doi.org/10.1007/ s00249-017-1233-7 Kohn F, Hauslage J, Hanke W (2017) Membrane fluidity changes, a basic mechanism of interaction of gravity with cells? Microgravity Sci Technol. https://doi.org/10.1007/s12217-017-9552-y Kordyum EL, Neduhka OM, Grakhov VP, Melnik AK, Vorbyova TM, Klimeko OM, Zhupanov IV (2015) Study of the influence of simulated microgravity on the cytoplasmic membrane lipid bilayer of plant cells. Kosm Nauka Tehnol 21:40–47 Lacowicz JR (2006) Principles in fluorescence spectroscopy. Springer, New York, NY Layne CS, Spooner BS (1990) EMG analysis of human postural during parabolic flight microgravity episodes. Aviat Space Environ Med 6:994–998 Leitgeb B, Szekeres A, Manczinger L, Vagvölgyi C, Kredics L (2007) The history of alamethicin: A review of the most extensively studied peptaibol. Chem Biodivers 4:1027–1051 Mallipattu SK, Haidekker MA, Von Dassow P, Latz M, Frangos J (2002) Evidence for shearinduced increase in membrane fluidity in the dinoflagellate Lingulodinium polyedrum. J Comp Physiol 188:409–416 Martins-Ferreira H, de Oliveira-Castro GD (1966) Light scattering changes accompanying spreading depression in isolated retina. J Neurophysiol 29:715–726 Meissner K, Hanke W (2002) Patch clamp experiments under microgravity. J Grav Physiol 9 (1):377–378 Meissner K, Hanke W (2005) Action potential properties are gravity dependent. Microgravity Sci Technol 17(2):38–43 Murray JD (2002) Mathematical biology. An introduction. Springer, New York Neef M, Ecke M, Hampp R (2015) Real-time recording of cytosolic calcium levels in Arabidopsis thaliana cell cultures during parabolic flights. Microgravity Sci Technol 27:305–312 Novespace (2009) A300 Zero-G rules and guidelines, RG-2009-2 Pandis C, Metastasio A, Mastrandrea F (2009) Effects of gravity on ulnar nerve latency of activation. http://eea.spaceflight.esa.int/attachments/parabolicflights/ID49661e9539c5f.pdf Pandolfi C, Masi E, Yoigt B, Mugnai S, Volkmann D, Mancuso S (2014) Gravity affects the closure of the trap in Dionaea muscipula. BioMed Res Int. https://doi.org/10.1155/2014/964203 Richard S, Henggeler D, Ille F, Vadrucci Beck S, Moeckli M, Forster IC, Franco-Obregón A, Egli M (2012) A semi-automated electrophysiology system for recording from Xenopus Oocytes under microgravity conditions. Microgravity Sci Technol 24(4):237–244. https://doi.org/10. 1007/s12217-012-9307-8 Rüegg DG, Kakebeeke TH, Studer LM (2000) Einfluss der Schwerkraft auf die Fortleitungsgeschwindigkeit von Muskel-Aktionspotentialen. In: Kelle H, Sahm PR (eds) Bilanzsymposium Forschung unter Weltraumbedingungen. WPF, Aachen, pp 752–759 Sagués F, Epstein IR (2003) Nonlinear chemical dynamics. Dalton Trans 7:1201–1217 Sieber M, Hanke W, Kohn FPM (2014) Modification of membrane fluidity by gravity. Open J Biophys 4(4):105–111 Sieber M, Kaltenbach S, Hanke W, Kohn F (2016) Conductance and capacity of plain lipid membranes under conditions of variable gravity. J Biomed Sci Eng 9:361–366 Singer SJ, Nicholson GL (1972) The fluid mosaic model of the structure of cell membranes. Science 175(4023):720–731 Tabony J (2006) Microtubules viewed as molecular ant colonies. Biol Cell 98:603–617 Tasaki I (2004) On the conduction velocity of non-myelinated nerve fibers. J Integr Neurosci 3:115–124 Torchilin VP, Weissig V (2003) Liposomes: a practical approaches. Oxford University Press, New York

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Vaquer S, Cuyas E, Rabadan A, Gonzale A, Fenollosa F, de la Torre R (2014) Active membrane drug transport in microgravity: a validation study using an ABC transporter model. F1000Research 3:201. https://doi.org/10.12688/f1000research.4909.1 Volinsky R, Kolusheva S, Berman A, Jelinek R (2004) Microscopic visualization of alamethicin incorporation into model membrane monolayers. Langmuir 20:11084–11091 Wiedemann M, Hanke W (2002) Gravity sensing in the central nervous system. J Grav Physiol 9 (1):43–44 Wiedemann M, Rahmann H, Hanke W (2003) Gravitational impact on ion channels incorporated into planar lipid bilayers. In: Tien HT, Ottova A (eds) Planar lipid bilayers and their applications. Elsevier Sciences, Amsterdam, pp 669–698 Wiedemann M, Kohn FPM, Rösner H, Hanke WRL (2011) Self-organization and pattern-formation in neuronal systems under conditions of variable gravity. Springer, Berlin. isbn:978-3-64214471-4 Wolfram S (2002) A new kind of science. Wolfram Media, Champaign, IL Woolley GA, Wallace BA (1992) Model ion channels: gramicidin and alamethicin. J Membr Biol 129(2):109–136 Zaikin AN, Zhabotinsky AM (1970) Concentration wave propagation in two-dimensional liquidphase self-oscillating system. Nature 225:535–537 Zanello LP, Aztiria E, Antollini A, Barrantes FJ (1996) Nicotinic acetylcholine receptor channels are influenced by the physical state of their membrane environment. Biophys J 70:2155–2164 Zhao H, Lappalainen P (2012) A simple guide to biochemical approaches for analyzing proteinlipid interactions. Mol Biol Cell 23(15):2823–2830. https://doi.org/10.1091/mbc.e11-07-0645

Chapter 2

Interaction of Gravity with Cell Metabolism

Abstract Plants orient their organs, explore, and adapt to their environment mainly by sensing light and the direction of gravity. Some theories exist about gravity sensing including as a starting point the presence of dense sedimentable particles in specialized gravity-sensing cell types or protoplast-pressure phenomena inducing a cascade of biophysical and biochemical events that finally transform the directional information into gravioriented growth. However, apart from the directional information, plant cells show various more general gravity effects like changes in membrane-located processes and changes in gene expression, protein expression, and protein modulation as well as metabolic consequences in response to altered gravity conditions. In the following, mainly based on data from callus cultures of A. thaliana, we summarize the present knowledge in the field of gravityaffected cell metabolism, especially related to Ca2+ and hydrogen peroxide signaling. Keywords Arabidopsis thaliana · Gene expression · Metabolic gravity response · Plants · Protein expression · Protein modulation · Secondary messengers

2.1

Introduction

Light and gravity are important environmental factors that control the development and growth of plants. With regard to gravity, this is quite obvious due to the spatial orientation of shoots, main roots, and first-order secondary roots. Investigations into the gravitational sensing of roots of higher plants give evidence for a cascade of biophysical and biochemical responses (Perera et al. 2001). For the initiation of such cascades, there are primarily two hypotheses. These are either based on the sedimentation of particles with high density such as starch-containing amyloplast statoliths (Sack 1991; Kiss 2000) or protoplast-pressure phenomena. The latter hypothesis suggests a perception of differences in pressure (reduced, increased) between the plasma membrane and the cell wall of opposing cell areas

© Springer Nature Switzerland AG 2018 W. Hanke et al., Gravitational Biology II, SpringerBriefs in Space Life Sciences, https://doi.org/10.1007/978-3-030-00596-2_2

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with the consequence of changed fluxes through mechano-sensitive ion channels (Staves 1997). Such a way of perception should function in any plant cell. It is also assumed that the actin cytoskeleton is included in response to altered gravity (Sievers 1991; Baluska and Hasenstein 1997; Rosen et al. 1999; Perbal and Driss-Ecole 2003; Soga et al. 2008). There is data, however, which indicates that at least in certain cell types, such as Chara rhizoids, actomyosin is more involved in fine tuning of responses than in gravity perception itself (Limbach et al. 2005; Braun and Limbach 2006). At the end of signal perception and transduction, there are metabolic responses, which help the cell to adjust to new gravitational steady states. These have been identified for primary, energy, and secondary metabolism (Obenland and Brown 1994; Hoffmann et al. 1996; Hampp et al. 1997). Model systems which are primarily investigated are Arabidopsis and rice. For Arabidopsis thaliana with its small genome, large data banks exist, which support expression studies for both genes and proteins. First reports about the long-term effect of microgravity (μg) on whole plants have been presented with material exposed on the International Space Station (ISS; Kiss et al. 2009; Pyle et al. 2001). A considerable number of gene transcripts were altered in amount including those involved in the jasmonic acid pathway. Downregulated genes included one coding for a peroxidase. Other ISS-related studies with dwarf wheat were not able to detect alterations in gene expression in 24-day-old leaves (Stutte et al. 2006). Changes in gene expression due to missing earth gravitation can be very fast. Callus cultures of Arabidopsis thaliana (A.t.) responded within minutes of microgravity as obtained by sounding rockets (Martzivanou et al. 2006). The impact of microgravity on gene expression results from perceived and transmitted signals (see above for gravity sensing). Independent of the external signal and its perception, environmental changes are transmitted by a transient increase of the intracellular calcium (Ca2+) concentration. This has also been shown for changes in the gravity vector (Toyota et al. 2008). Another trigger is the production of reactive oxygen species (ROS). They are no longer considered only as protectants against invading pathogens. ROS such as hydrogen peroxide have been shown to be also important in cellular signaling, and a “ROS gene network” has been suggested (Dodd et al. 2010; Neill et al. 2002; Miller et al. 2008). There are also reports about a close interrelationship between the hydrogen peroxide producing NADPH oxidase, hydrogen peroxide, and Ca2+ (Wong et al. 2007; Takeda et al. 2008). Here, we mainly report about studies with callus cultures of A. thaliana. These deal with alterations in the pools of these secondary messengers, related gene and protein expression, as well as protein modification, and integrate metabolic alterations.

2.2 Methods

2.2

35

Methods

2.2.1

Opportunities for Exposure

Centrifugation (hypergravity) and clinorotation (2-D), or random positioning of samples (RPM), are well-established methods to investigate effects of gravistimulation in comparison with 1 g. In the case of clinorotation, the idea is that a constant stimulus acting similarly on all parts of a plant/tissue/cell could simulate microgravity. RPM treatments result in randomly disturbed cells, possibly prohibiting specific responses and thus simulating weightlessness. Magnetic levitation is a not so widely used technique, exploiting a strong magnetic field to counteract the earth’s gravitational field and thus possibly simulating weightlessness.

2.2.1.1

Centrifugation

Application of hypergravity was done by centrifugation of Petri dishes (Fig. 2.1, Babbick et al. 2005; Martzivanou and Hampp 2003). After centrifugation, cells were scraped off and directly frozen in liquid nitrogen. The fluorometric analyses were performed with the centrifuge at the ZARM Institute in Bremen (http://www.zarm. uni-bremen.de/menu/facilities/centrifuge/; Neef et al. 2015). Fig. 2.1 Petri dish centrifuge (University of Tübingen, Germany)

36

2.2.1.2

2 Interaction of Gravity with Cell Metabolism

Clinorotation

Clinorotation was performed at 60 rpm with tubes having an internal diameter of 10 mm. This arrangement resulted in a maximal gravitational force of 0.0016 g (Hemmersbach, DLR, personal communication). Acceleration forces of 0.5 g and less were simulated with rotating Petri dishes (Fengler et al. 2015a, b).

2.2.1.3

Random Positioning

For random positioning, cultures were prepared on Petri dishes as described above for centrifugation. The dishes were then fixed in the center of the inner of the two connected frames. The frames were rotating in a random, autonomous way at an angular velocity of 60
s1 (Walther et al. 1998). For details of the different procedures, see also Babbick et al. (2007).

2.2.1.4

Magnetic Levitation

A superconducting magnet with a closed-cycle liquid helium cooling system (Oxford Instruments, Abingdon, UK), capable of generating a very high magnetic field gradient, was used as another means to investigate the effects of simulated weightlessness on gene expression in Arabidopsis callus. For levitation, simulated weightlessness (sim 0 g) is defined as the condition, in which the magnetic force balances the weight of gravity of an object with certain caveats (Catherall et al. 2005). Cells (0.5 g f. wt.) were dispensed onto 3 ml aliquots of agar-solidified culture medium in 25 ml capacity, 2.7 cm diameter, screw-capped culture tubes (Sarstedt, Leicester, UK). After 7 days of culture, the tubes containing the samples were held at specific positions in the magnet bore in the dark using a purpose-built Perspex cylinder. The cells were exposed to 0 g and 1 g inside the magnet in the presence of a strong magnetic field. Cells at the 1 g position in the magnet were used as a control for the effect of the magnetic field alone (16.5 T). A constant temperature of 24  1
C was maintained in the magnet bore during the experimental period. Additional 1 g control cultures were placed in a temperature-controlled growth chamber in the dark outside the magnetic field.

2.2.1.5

Space Shuttle and Satellite

An extended period of microgravity was made available by the German-Chinese satellite mission Shenzhou 8. The respective flight and ground hardware (SIMBOX) were developed by Astrium GmbH (now Airbus D&S, Friedrichshafen, Germany) under a contract of the German Space Administration (DLR). The incubator contained a platform and a 1 g reference centrifuge. One out of two cultivation

2.2 Methods

37

Fig. 2.2 Fully automated Type V plant cell cultivation unit (without cover) as used for Shenzhou 8

units (EUEs—experiment unique equipment) was fixed on the experimental platform and experienced microgravity. The second unit provided the in-flight reference by experiencing 1 g during the microgravity phase. Each module consisted of two cultivation chambers (Fig. 2.2). In order to prevent anaerobiosis, samples were fixed after 5 days by the injection of the fixative RNAlater®. Finally, the samples were harvested and stored at 4
C (Fengler et al. 2015a, b).

2.2.1.6

Sounding Rockets

This experimental setup was based on syringes. One of two 2 ml syringes was loaded with 90 mg callus culture and 30 μl liquid culture medium. A bead sitting in the tip of the syringe closed the vessel. Via an adaptor this syringe was connected to the other one, which contained the stop solution (1 ml RNAlater; Qiagen, Germany; Fig. 2.3). The syringes were placed in a module, sitting in the payload of the rocket. Total RNA from the cells was fixed at a defined time (i.e., 5 and 10 min μg) by pistonactuated closure of the syringe, containing the stop solution. For each time point, 8 incubation/quenching units were used in parallel. 1 g control samples were treated exactly the same way outside the payload. After recovery of the sample module by helicopter (within 1 h after launch), the RNAlater-treated samples were transferred into Eppendorf cups and kept between 4–8
C until RNA extraction.

2.2.1.7

Parabolic Flights: Hyper-g, Partial g, and Microgravity

A typical parabola is illustrated in Fig. 2.4. The flights (Airbus A300, Novespace, France) took place between 6000 and 8500 m a.s.l. Parabolas consist of several phases: during ascend, the aircraft accelerates with 1.8 g (directed toward the ground

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2 Interaction of Gravity with Cell Metabolism

Fig. 2.3 Syringe-based quenching system. One syringe containing the cell culture is connected by a Teflon tube to a second syringe containing the quenching agent. The syringe with the quenching solution was sealed with a bead. When the cell culture syringe was activated at a given time, the bead was pushed out, and the quenching agent was injected into the culture tube (used for sounding rocket experiments)

Fig. 2.4 Schematic representation of a parabolic flight maneuver. The numbers indicate manual sampling events for transcript and proteome analyses (modified after Hausmann et al. 2014)

floor) in an angle of about 47
. After about 20 s, the pilots strongly reduce the thrust, and the plane falls along the top of the parabola by its residual speed, with only some minor thrust to compensate for the air drag. During this flight phase, microgravity in a range of 102 g is provided for 22 s. Finally, within 20 s, the plane returns to a normal horizontal flight, causing an acceleration level of 1.8 g again. At 4 time

2.2 Methods

39

points (indicated by numbers 1–4 in Fig. 2.4), samples were taken (e.g., for extraction of RNA and proteins) in cases, when no continuous measurements were made (e.g., fluorescence). By adapting the flight angle and the overall velocity of the plane, the cells were additionally exposed to 0.38 and 0.16 g (Mars and Moon gravity during a Joint European Partial-G Parabolic Flight campaign).

2.2.2

Plant Material

Cell suspension cultures and calli were generated from leaves of Arabidopsis thaliana (cv. Columbia) plants grown under sterile conditions as detailed elsewhere (Barjaktarović et al. 2007). Calli with a diameter of about 1 mm were obtained after 1 week of growth and were used for the experiments.

2.2.3

Determination of Key Metabolites

Determination of ATP, ADP, and AMP from HClO4 extracts was by luminescence according to Hampp et al. (1985, 1997). Quantitation of triphosphopyridine nucleotides was done by a cycling technique (BioVision, BioCat, Heidelberg, Germany; Hausmann et al. 2014). The determination of fructose 2,6-bisphosphate (F26BP) was carried out according to Steingraber et al. (1988).

2.2.4

Metabolic Labeling with (14C)-Glucose

During the TEXUS 37 sounding rocket campaign, we had the chance to perform radioactive labeling experiments. For this purpose, cell suspensions were incubated at about 24
C in one of three 2 ml syringes, which were connected to each other via a T-piece (Fig. 2.5). The incubation with (14C)-labeled glucose was started by closing the respective syringe and subsequent injection of the solution (incubation medium containing 1 mM glucose þ 0.2 MBq of 14C; specific activity, 12 GBq mmol1) into the syringe containing the cell suspension. Labeling was terminated by the injection of ethanol (final concentration approx. 70%) from the third syringe (Fig. 2.5). Separation of metabolites contained in 2 μl aliquots was by thin-layer chromatography (TLC) on cellulose plates (Merck, Darmstadt, Germany) according to Feige et al. (1969). The spots were made visible by exposure to X-ray film (Amersham, Braunschweig, Germany) for about 3 months

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Fig. 2.5 Incubation of cell cultures for labeling experiments. Right (third syringe), labelled glucose; middle (second syringe), cell suspension; left (1st syringe), EtOH. The three syringes are connected via channels in a Perspex block and numbers 1 and 3 sealed by small beads. Syringes 1 and 3 are actuated in a time-dependent manner

at room temperature. In order to verify the different compounds, defined mixtures of the putative metabolites were run in parallel (þ/ cell extracts).

2.2.5

Real-Time Analysis of Ca2+ and Hydrogen Peroxide

Seeds of A. thaliana plants expressing Yellow Cameleon were kindly provided by K. Schumacher/M. Krebs (University of Heidelberg, Germany). For construct details, see Allen et al. (1999) and Miyawaki et al. (1999). cHyPer cell culture lines, generated from shoots of 6-day-old transgenic seedlings (Costa et al. 2010), were obtained from A. Costa (University of Padua, Italy). Fluorescence of samples was recorded by means of a microplate reader (POLARstar Optima; BMG, Germany; Hausmann et al. 2014).

2.2.6

Gene and Protein Expression

Isolation of total RNA and protein, and microarray analysis (Affymetrix GeneChip) is detailed in Barjaktarović et al. (2007, 2009a, b), Neef et al. (2013a), and Hausmann et al. (2014).

2.3 Results

2.3 2.3.1

41

Results Metabolism

A consistent observation regarding metabolism of plants grown in microgravity or under horizontal clinorotation are changes in carbon metabolism. This can be a decrease (e.g., Obenland and Brown 1994; leaves exposed to microgravity), as well as an increase in starch accumulation (cell cultures; Wang et al. 2006). Since the carbon metabolism is closely linked to energy metabolism, we assumed and finally could show that changes in gravity have an impact on the cellular energy metabolism (Hampp et al. 1997). First attempts to study metabolic responses to changes in gravity were made during preparation (sounding rocket experiments) and execution of the D2 mission (1993). In these experiments, we studied pool sizes of adenylates, pyridine nucleotides, and a metabolite regulator of glycolysis, F26BP. Exposure of cell (protoplast) suspensions to microgravity on a short-term basis (sounding rocket flights) resulted in changes in the cellular energy and redox state within a few minutes of exposure (Hampp et al. 1997); i.e., the ratio of ATP/ADP started to fluctuate considerably reaching values of up to 2 as compared to 1 in 1 g controls. The decrease in NAD+ indicated an increase of the NADH pool, while the rise of NADP+ could be related to a decrease of NADPH (this is especially interesting with regard to the stress-related formation of reactive oxygen species as is discussed later). Centrifugation experiments also showed an increased NADH/ NAD ratio in A.t. callus cells exposed for 1 h to between 6 g and 9 g (Fig. 2.6). Finally, random positioning resulted in an extended increase in this ratio between approx. 1 and 16 h of exposure (Maier et al. 2003).

Fru2,6bisP (pmol·106 protoplasts)

200 180 160 140 120 100 80 60 40 μg

lift off

20 0 0

5

10

15 20 Sample number

25

30

35

Fig. 2.6 Ratio of NADH/NAD in A. thaliana cell cultures during a sounding rocket flight (TEXUS). Samples were quenched every 20 s. The μg phase ended around sample 26 (Hampp et al. 1992)

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2 Interaction of Gravity with Cell Metabolism

In addition, altered pool sizes of a regulator of plant cell glycolysis, fructose-2,6 bisphosphate (F26BP), were found. Under sounding rocket microgravity, this compound increased in pool size (Hampp et al. 1997; Fig. 2.6). As elevated levels of F26BP favor glycolysis, we assumed that this could be related to an enhanced use of intermediates of glycolysis for either respiratory ATP production or de novo synthesis of proteins. Another indication that changes in the effective gravitational field affect glycolysis comes from hyper-g (7 g) and random positioning experiments (Maier et al. 2003). Here, increased ratios of the glycolysis intermediates, pyruvate/phosphoenolpyruvate (PEP), were found (Fig. 2.7). These suggest a piling up of pyruvate at an increased consumption of PEP. Together with an increased PEP carboxylase expression (PEPC; see below; Maier et al. 2003), this was taken as indicative for stress compensation (increased rates of anaplerosis related to protein synthesis). The enzyme phosphoenolpyruvate carboxylase (PEPC) is a key component of anaplerosis. Due to a limited availability of flight opportunities, we thus focused on centrifugation experiments. Hypergravity resulted in a transient increase of PEPC activity after 1 h exposure to g-forces between 6 and 10 (Fig. 2.8). This was obviously due to de novo synthesis, as both enzyme-specific protein (Western blot; Fig. 2.9) and the amount of the respective transcript increased (Maier et al. 2003; Fig. 2.10). Posttranslational modulation was not involved, as the activation (¼ phosphorylation) state of the enzyme was not altered. Pool sizes of pyruvate (pyr), phosphoenolpyruvate (PEP), and the ratio of NADH/NAD suggested an inhibition of the pyruvate dehydrogenase complex in favor of conversion of PEP to oxaloacetate, which is the precursor for the synthesis of amino acids of the aspartate family.

Pyruvate and PEP - HyperG 500

pmol/mg dw

400 300 200 100 Pyruvate

PEP

0 0

2

4

6 G-Force [1h]

8

10

12

Fig. 2.7 Contents of pyruvate and phosphoenolpyruvate (PEP) in A. thaliana cell cultures which were treated for 1 h at different g-forces

2.3 Results

43

Fig. 2.8 Activity of phosphoenolpyruvate carboxylase (PEPC) from A. thaliana cell cultures upon centrifugation for 1 h at different g-forces

Fig. 2.9 Western blot with phosphoenolpyruvate carboxylase-specific antibodies. A. thaliana cell cultures were exposed for 1 h at different g-forces

Fig. 2.10 Phosphoenolpyruvate carboxylase-transcript, detected by RT-PCR in A. thaliana cell cultures which were exposed for 1 h at different g-forces. Total RNA was used as a reference

Clinorotation of the cells also caused a transient increase in PEPC activity (between 30 min and 16 h), which was not mirrored by changes in the amounts of the respective transcript. Under clinorotation, the enzyme is probably regulated by posttranslational modification. Independent of the way of regulation, pool sizes of pyr, PEP, and the ratio of NADH/NAD changed as shown for hypergravity conditions. This suggests a similar response but triggered in a different way.

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2 Interaction of Gravity with Cell Metabolism

Fig. 2.11 Autoradiographs of the fixation pattern after incubation with (14C)-labeled glucose of the 1 g control (a) and after 6 min of microgravity (TEXUS) (b). The arrows indicate three spots which are increased after exposure of cell cultures of A. thaliana to μg. DHAP dihydroxyacetone phosphate; Fru fructose; Glu glucose; P phosphate; Suc sucrose

In addition, labeling patterns after feeding of (14C)glucose were determined. Both labeling experiments and RNA analysis of samples exposed to sounding rocket microgravity (TEXUS) showed fast responses of exposed A. thaliana cell suspensions. With regard to labeling, we detected an increase in the abundance of three metabolites of the organic/amino acid fraction after 6 min of microgravity. The nature of these intermediates could not be resolved, because cochromatography with 53 reference metabolites did not reveal identical Rf values (Fig. 2.11). PEPC expression was not altered within this time frame.

2.3.2

Secondary Messengers: Calcium and Hydrogen Peroxide

In plant cells, calcium (Ca2+) ions play a key role as an intercellular second messenger in response to a variety of environmental stimuli such as light, touch, pathogenic elicitors, plant hormones, high salinity, cold, and drought (Dodd et al. 2010; Sarwat et al. 2013). Spatially and temporally distinct changes of the cytosolic Ca2+ concentration are thought to constitute a unique signal for its inducing stimulus. Such stimulusspecific spikes in the cellular Ca2+ concentration are also known as Ca2+ signatures and result usually from two opposing responses: Ca2+ influx through channels or Ca2+ efflux through pumps (Tuteja and Mahajan 2007). Up to now, three different types of cytosolic Ca2+ signatures are described in the literature: oscillatory (e.g., after cold, ABA, or nod-factor treatment); biphasic, with a large, rapid first increase and a second smaller but sustained increase (e.g., in response to bending); and monophasic with a

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single cytosolic Ca2+ increase (e.g., due to red/blue light, salinity, heat, and touch; Batistic and Kudla 2012). Also, gravity sensing is thought to be transmitted by changes in the concentration of cytosolic Ca2+ (Poovaiah et al. 1987, 2002; Plieth and Trewavas 2002; Gehring et al. 1990; Gilroy et al. 1993; Fasano et al. 2002; Correll et al. 2013). More recently, visualization and analysis of intracellular Ca2+ dynamics in living plants were revolutionized through the introduction of genetically encoded Ca2+ sensors (e.g., aequorin or GFP-based calcium probes like YC3.6; Knight et al. 1997; Allen et al. 1999; Nagai et al. 2004). Toyota and coworkers used A. thaliana seedlings expressing cytoplasma-targeted apoaequorin for their analyses and described a biphasic cytosolic Ca2+ increase during parabolic flights, which included a plant rotation in addition to the gravistimulation (Toyota et al. 2013; Toyota and Gilroy 2013). Without rotation, a delayed and sustained monophasic cytosolic Ca2+ increase was observed (Toyota et al. 2013). For our analyses, we used a YC3.6 construct, which possesses reduced pH sensitivity and, owing to the UBQ10 promoter, exhibits a high rate of expression in A. thaliana (Krebs et al. 2012; Behera et al. 2013). With this construct we found cytosolic Ca2+ fluctuations which run parallel to gravitational changes (hypergravity and microgravity) during parabolic flights (Fig. 2.12). In order to get an idea about the source of the μg-related increase of cytosolic Ca2+, we used a set of known and established Ca2+ inhibitors/antagonists to affect either the Ca2+ influx into or the Ca2+ efflux out of the cell. The data show a fast rise in cytosolic Ca2+ upon the onset of microgravity and indicate that cytosolic Ca2+ is mainly of extracellular origin. In contrast, exposure to hypergravity resulted in decline of the cytosolic amount of Ca2+ starting from between 3 g and 4 g (Fig. 2.13; Neef et al. 2016). Another trigger for the transmission of environmental changes is the production of reactive oxygen species (ROS). They are no longer considered only as protectants against invading pathogens. ROS such as hydrogen peroxide have been shown to be 1.055 Ca concentration (mM)

1.05 1.045 1.04 1.035 1.03 1.025 1.02 1.015 1.01

1 g control

1.8 g

mg

Fig. 2.12 Intracellular Ca2+ concentration depending on gravity. The values are means of 24 parabolas SD (n ¼ 24). The control was set as 100% [1.03 μM; mean of all control readings (1 g)]. The differences between 1.8 g and microgravity, as well as between 1 g and microgravity, are highly significant according to Tukey’s multiple comparison of means (***P < 0.001). There was no significant difference between in-flight 1 g and initial 1.8 g (Hausmann et al. 2014)

Fig. 2.13 Ca2+ transients in different intracellular compartments under hypergravity. Primary y-axis, fluorescence ratio (530/480 nm; black line); secondary yaxis, acceleration (g gray dotted line); x-axis, time (minutes); (a) NES (cytosol and nucleus), (b, c) NLS (nucleus), (d) TP12 (tonoplast) (Neef et al. 2016). NES, NLS, TP12: compartment-specific expression of the cameleon construct (Neef et al. 2015)

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also important in cellular signaling, and a “ROS gene network” has been suggested (Dodd et al. 2010; Neill et al. 2002; Miller et al. 2008). There are also reports about a close interrelationship between the hydrogen peroxide producing NADPH oxidase, hydrogen peroxide, and Ca2+ (Wong et al. 2007; Takeda et al. 2008). For the determination of hydrogen peroxide, we employed cell cultures expressing the yellow fluorescent protein-based sensor HyPer (Belousov et al. 2006). This sensor is specific to this component of reactive oxygen species. For experimental details, see Neef et al. (2015). Here, we also found an increase in this ROS component upon lack of gravity (Fig. 2.14). Altogether, we evaluated about 60 parabolas during independent flights in 2011 and 2012. The responses of the cell cultures were more variable for hydrogen peroxide than for Ca2+. The high number of “non-responding” cultures (40%) could also be due to the lower sensitivity of the HyPer system. Hydrogen peroxides can be produced by NADPH oxidase, located in the plasma membrane. This enzyme is activated by Ca2+ and releases hydrogen peroxide to the apoplast (Wong et al. 2007). It is fueled by intracellular NADPH, which probably is generated by glucose 6P dehydrogenase (oxidative pentose phosphate cycle). If indeed significant amounts of hydrogen peroxide are formed during onset of microgravity, pool sizes of NADPH should respond. We thus assayed the amounts of NADPH and NADP. In this case samples were taken at the points indicated in Fig. 2.4 and quenched in either acid (NADP) or base (NADPH). The calculated ratios are given in Fig. 2.15. At 1 g (before acceleration), the ratio is around 1.2. At the onset of μg, the ratio drops (0.9) but rises during the μg phase, reaching nearly 1.7 at the end. At the end of the second 1.8 g phase, the ratio comes back to control values. Especially the behavior during microgravity, which is opposite to that of hydrogen peroxide, indicates some interdependence: the onset of hydrogen peroxide

Fig. 2.14 Amounts of hydrogen peroxide, shown as fluorescence emission ratio during a parabola (excitation ratio 480:420 nm/emission 530 nm). An increase in the ratio indicates an increased H2O2 content. The graph presents the typical behavior of a responsive callus. The dotted line gives the respective kinetics of the acceleration, ranging from